NOTCH1 Mutations in Aortic Stenosis: Association with Osteoprotegerin/RANK/RANKL

Abstract

Background. The NOTCH pathway is known to be important in the pathogenesis of calcific aortic valve disease, possibly through regulators of osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK), and its ligand (RANKL) system. The purpose of the present study was to search for possible associations between NOTCH1 gene mutations and circulating levels of OPG and soluble RANKL (sRANKL) in patients with aortic stenosis (AS). Methods. The study was performed on 61 patients with AS including 31 with bicuspid and 30 with tricuspid aortic valves. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Serum OPG and sRANKL levels were assessed. Results. In total, 6 genetic variants of the NOTCH1 gene including two new mutations were identified in the study group. In an age- and arterial hypertension-adjusted multivariable regression analysis, the serum OPG levels and the OPG/sRANKL ratio were correlated with NOTCH1 missense variants. All studied missense variants in NOTCH1 gene were found in Ca(2+)-binding EGF motif of the NOTCH extracellular domain bound to Delta-like 4. Conclusion. Our results suggest that the OPG/RANKL/RANK system might be directly influenced by genetic variants of NOTCH1 in aortic valve calcification.

Figure 1

Localization of missense mutations in NOTCH1. (a) DNA sequences, characterized by location of the missense mutations in three patients in comparison with the reference sequence of NOTCH1 gene (NG_007458.1). (b) Multiple sequence alignment of wild-type NOTCH1 protein (Uniprot ID: P46531) and mutated variants located in patients. EGF CA domain is a calcium-binding epidermal growth factor-like domain of NOTCH1. The amino acid residues in boxes are mutated positions.

Figure 2

Structure of NOTCH1 EGF CA domain. (a) The sequence alignment of human NOTCH1 EGF CA domain with rat NOTCH1 EGF CA domain of known crystal structure (PDB ID: 4XLW Chain A) visualized in Jalview 2.8.2 [11]. Highly conserved residues (conservation score = 11) are marked as “∗” and identical residues (conservation score = 10) are marked as “+.” The amino acids in red boxes are mutated positions. (b) Three-dimensional structure of EGF modules of the NOTCH extracellular domain bound to DLL4 [12] visualized by PyMol. Structures of proteins are shown in cartoon representation. Calcium ions are represented as gray spheres. β-D-glucose (BGC) and α-L-fucose (FUC) molecules are highlighted in yellow. Residues of interest E1305, D1267, and R1279 are marked as sticks in red color.

Figure 3

Box and whisker plots the distributions of serum OPG levels in patients with or without NOTCH1 missense variants: 7.9 (5.3, 9.4) and 5.3 (4.6, 7.2), P = 0.044, comparable.